The long term goal of the proposed research is to understand at a molecular and structural level how TRPV ion channel responses integrate external stimuli and cellular state. The TRPV channels are important in sensory and pain perception and in calcium homeostasis. At a molecular level, the large N- and C-terminal cytosolic domains of TRPV channels sense information about the cellular state - calcium and phosphoinositide levels, for example - to regulate channel sensitivity. We have recently identified a physiologically important multi-ligand binding site within the TRPV1-ARD that modulates TRPV1 channel sensitivity. This proposal expands on these recent results to address how interactions of small molecule and protein ligands with the TRPV N- and C-terminal cytosolic regions synergize to regulate channel sensitivity. TRPV ion channels are important drug targets and the results of the proposed structure and function studies of TRPV channels will enable new structure-based design strategies for drug development. The work can be divided in the following specific aims: Aim 1. To structurally characterize the regulatory ligand binding sites in the TRPV1 cytosolic regions: Our working model is that upon TRPV1 channel opening, as Ca2+ enters the cell, PIP2 is released from the TRPV1-CT and ATP from the TRPV1-ARD, such that Ca2+-bound CaM can crosslink the Nand C-termini of TRPV1, thereby inactivating the channel. We will use biochemical, structural and electrophysiological experiments to test this working model and address the functional relationship between the N- and C-terminal ligand binding sites in TRPV1. Aim 2. To characterize shared and unique regulatory interactions of the ARDs of TRPV channels: TRPV1 is the best studied of the six mammalian TRPV subfamily members. We take advantage of our success in preparing TRPV-ARDs from all family members to determine whether any other TRPV-ARD shares the ATP or CaM binding site found in TRPV1 and, if so, whether the ATP- or CaM-mediated channel regulation mechanisms are shared. We will also determine the structure of several unique TRPV-ARD interactions. The results will provide insights into the extent of conservation vs. specialization of the function and regulation of TRPV channels. Aim 3. To identify the structural determinants of capsaicin sensitivity in the TRPV1 channel: Ultimately, to understand how channel sensitivity is regulated, we also need to understand how the channel is activated. The goal of this aim is to pinpoint the regions within TRPV1 that bind capsaicin or change conformation upon channel activation, through a combination of mutagenesis, chemical modifications and electrophysiology. The capsaicin binding site is a prime location within the channel to target with potential pain relief drug compounds. Knowledge we gain about the capsaicin binding site and how capsaicin affects the conformation of the TRPV1 channel can therefore be applied to drug development, directly linking our results to healthcare research.